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Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus Macrops and T

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Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus Macrops and T toxins Article Venomics and Cellular Toxicity of Thai Pit Vipers (Trimeresurus macrops and T. hageni) Supeecha Kumkate 1, Lawan Chanhome 2 , Tipparat Thiangtrongjit 3, Jureeporn Noiphrom 4, Panithi Laoungboa 2, Orawan Khow 4, Taksa Vasaruchapong 2, Siravit Sitprija 1, Narongsak Chaiyabutr 2,* and Onrapak Reamtong 3,* 1 Department of Biology, Faculty of Science, Mahidol University, Ratchathewi, Bangkok 10400, Thailand; [email protected] (S.K.); [email protected] (S.S.) 2 Snake Farm, Queen Saovabha Memorial Institute, The Thai Red Cross Society, Pathumwan, Bangkok 10330, Thailand; [email protected] (L.C.); [email protected] (P.L.); [email protected] (T.V.) 3 Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok 10400, Thailand; [email protected] 4 Department of Research and Development, Queen Saovabha Memorial Institute, The Thai Red Cross Society, Pathumwan, Bangkok 10330, Thailand; [email protected] (J.N.); [email protected] (O.K.) * Correspondence: [email protected] (N.C.); [email protected] (O.R.) Received: 19 December 2019; Accepted: 13 January 2020; Published: 16 January 2020 Abstract: The two venomous pit vipers, Trimeresurus macrops and T. hageni, are distributed throughout Thailand, although their abundance varies among different areas. No species-specific antivenom is available for their bite victims, and the only recorded treatment method is a horse antivenom raised against T. albolabris crude venom. To facilitate assessment of the cross-reactivity of heterologous antivenoms, protein profiles of T. macrops and T. hageni venoms were explored using mass-spectrometry- based proteomics. The results show that 185 and 216 proteins were identified from T. macrops and T. hageni venoms, respectively. Two major protein components in T. macrops and T. hageni venoms were snake venom serine protease and metalloproteinase. The toxicity of the venoms on human monocytes and skin fibroblasts was analyzed, and both showed a greater cytotoxic effect on fibroblasts than monocytic cells, with toxicity occurring in a dose-dependent rather than a time-dependent manner. Exploring the protein composition of snake venom leads to a better understanding of the envenoming of prey. Moreover, knowledge of pit viper venomics facilitates the selection of the optimum heterologous antivenoms for treating bite victims. Keywords: Trimeresurus macrops; Trimeresurus hageni; pit vipers; snake venom proteomics; cytotoxicity; U937 monocytes; fibroblasts Key Contribution: This study revealed for the first time the venomic proteomes of the large-eyed pit viper (Trimeresurus macrops) and Hagen’s pit viper (T. hageni). Both snakes are widespread across Thailand and Southeast Asia. We quantitatively analyzed different protein clusters from these venoms. The cellular toxicity of the venoms on human fibroblasts and monocytes was also investigated. 1. Introduction Venomous pit vipers are snakes of the Crotalinae subfamily, characterized by two movable fangs and heat-sensing pit organs located bilaterally between the eye and nostril. Trimeresurus is a prominent pit viper genus and comprises the greatest number of known species [1]. Trimeresurus snakes are endemic to Asia; they are widely distributed, ranging from deserts to rainforests and in terrestrial, arboreal, and aquatic habitats. Trimeresurus bite victims have been reported in several geographic Toxins 2020, 12, 54; doi:10.3390/toxins12010054 www.mdpi.com/journal/toxins Toxins 2020, 12, 54 2 of 13 regions, including Lao PDR [2], Hong Kong [3], Taiwan [4], Thailand [5], China [6], Sri Lanka [7], and Japan [8]. In addition, green pit vipers were responsible for 58% of snakebites reported in Vietnam in 2017 [9]. Trimeresurus venom varies in toxicity between species; prolonged clotting time is a significant symptom observed in humans [10], and tissue damage and hematotoxicity in bite victims have also been reported [11,12]. Since there is no species-specific antivenom available for Trimeresurus, except T. albolabris, the only treatment available for bite cases has been a hetero-specific antivenom [13]. Antivenoms raised in horses are the most common therapeutic agents for snakebite treatment; however, they can cause several side effects, such as anaphylactic shock and serum sickness [14]. Moreover, preparation of antivenom from horse blood is laborious and time-consuming with a low production yield [15]. According to proteomics studies, each specific venom contains a unique variety of toxins [16]. To date, T. insularis (Indonesian), T. borneensis (Borneo), T. stejnegeri (Taiwan), T. puniceus (Java), T. purpureomaculatus (Thailand), T. gramineus (India), T. nebularis (Malaysia), and T. alborabris (Thailand) have been investigated for their venom constituents [17–19]. However, there is no reported information on the venomic protein profile of the Southeast Asia endemic species T. macrops and T. hageni. The large-eyed pit viper T. macrops can be distinguished from other green pit vipers by its relatively large eyes (Figure1a). T. macrops bites frequently cause severe tissue damage in humans with the symptoms ranging from local swelling to severe systemic bleeding [20]. Its venom has a long half-life and can be retained within the human body for more than 14 days [21]. T. hageni, known as Hagen’s pit viper is also endemic to Southeast Asia (Figure1b); however, there are only a few reports of the major symptoms of T. hageni venom. In addition, no T. macrops or T. hageni species-specific antivenoms are available. The antivenom raised from T. albolabris is currently used for neutralizing T. macrops and T. hageni venoms. In our study, a comparative proteomics approach was applied to the study of T. macrops and T. hageni venom protein composition. Moreover, we assessed the cytotoxicity of T. macrops and T. hageni venoms on monocytic cells (U937 cells) and skin fibroblasts (CRL-1474 cells) to clarify the effects on cellular physiology. The findings facilitate the analysis of cross-reactivity between these snake toxins and the available antivenoms. The identified toxins may be used for the development of inhibitory or neutralizing agents using molecular techniques to improve snakebite treatment. In addition, some proteins with beneficial activities could be further developed as novel pharmacological agents for human disease. Figure 1. Two prominent species of Trimeresurus snakes in Thailand. Adult large-eyed pit viper (T. macrops) with its noticeably large eyes (a) and the Hagen’s pit viper (T. hageni) perching on a tree branch (b). 2. Results 2.1. Proteomics Analysis of T. macrops and T. hageni Venom After preparing venom from T. macrops and T. hageni, the proteins were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure2). Bands at 15, 25, 35, 50, and Toxins 2020, 12, 54 3 of 13 55 kDa were the most abundant in T. macrops venom. Whereas, bands at 15, 16, 20, 22, 32, 55, and 66 kDa were the most intense in T. hageni venom. Each gel lane was excised into 10 pieces. Peptides were extracted from the gel by in-gel digestion and further subjected to liquid chromatography–mass spectrometry (LC-MS/MS) analysis. After MASCOT searching against NCBI (Taxonomy: Chordata), the results revealed that the T. macrops and T. hageni venoms contained 185 and 216 proteins, respectively (Supplementary Table S1). The identified proteins were classified according to their gene ontology, including molecular function, biological process, and cellular component terms (Table1). Figure 2. Coomassie blue-stained 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis of T. macrops and T. hageni venoms (30 µg) under reducing conditions. Table 1. Gene ontology classification of Trimeresurus macrops and T. hageni crude venom proteins. % of Protein Components Gene Ontology T. macrops T. hageni Molecular Function binding (GO:0005488) 12.5 33.3 catalytic activity (GO:0003824) 75.0 44.4 structural molecule activity (GO:0005198) 12.5 - molecular function regulator (GO:0098772) - 22.2 Biological Process biological regulation (GO:0065007) 36.4 20.0 cellular process (GO:0009987) 27.3 26.7 metabolic process (GO:0008152) 9.1 6.7 rhythmic process (GO:0048511) 27.3 20.0 immune system process (GO:0002376) - 6.7 response to stimulus (GO:0050896) - 6.7 localization (GO:0051179) - 13.3 Cellular component cell (GO:0005623) 25.0 33.3 extracellular region (GO:0005576) 12.5 16.7 membrane (GO:0016020) 12.5 - organelle (GO:0043226) 37.5 50.0 protein-containing complex (GO:0032991) 12.5 - Toxins 2020, 12, 54 4 of 13 In terms of molecular function, most T. macrops (75%) and T. hageni (44.4%) venom proteins were involved in catalytic activity. Structural molecule activity proteins were observed only in T. macrops venom and represented 12.5% of the proteins. Whereas, molecular function regulators comprised 22.2% of T. hageni venom proteins. In terms of biological processes, proteins involved in biological regulation and cellular processes were the largest classes present in T. macrops (36.4%) and T. hageni (26.7%) venoms, respectively. While immune system process, response to stimulus, and localization proteins were found only in T. hageni venom. In terms of cellular processes, organelle proteins were found in both T. macrops (37.5%) and T. hageni (50%) venoms. Whereas, membrane and protein-containing complex molecules were presented only in T. macrops venom. Phospholipase A2 (PLA2), snake venom serine protease (SVSP), cysteine-rich secretory, snake venom metalloproteinase (SVMP), disintegrin, L-amino acid oxidase, and C-type lectin were common to the two snake venoms. These protein families contribute to the phenotypic effects of venom on victims. Therefore, all identified proteins were also classified according to the common properties of snake venom, as shown in Figure3. PLA2, SVSP, cysteine-rich secretory, SVMP, and disintegrin were more abundant in T. macrops venom. Whereas, L-amino acid oxidase and C-type lectin were more abundant in T. hageni venom. Due to protein semi-quantification, the exponentially modified protein abundance index (emPAI) was used to estimate the amount of proteins. The 20 most abundant proteins in T.
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